Leica Confocal Buyers Guide

Buyer's Guide: Confocal Microscopes for Life Science Discovery Bringing focus to your decision-making process so you make the best choice for you and your lab. Budget Your Purchase ›› Step 4. Learn About Reputation & Support ›› Step 5.Inside… ›› Step 1. Know the Terms ›› Step 6. Make a Confident Decision . Understand the Options ›› Step 3. Evaluate Your Research Needs ›› Step 2. Choosing the right confocal microscope is one of the most important decisions you will make for your lab. In this guide. it has become the standard for fluorescence microscopy. Some labs are finding it beneficial to purchase their own confocal microscope rather than continuing to share one at a core lab. Today. structures. you’ll learn the key considerations for each step of the purchasing process. and tissues — visualization that goes well beyond what can be seen through conventional widefield microscopy. oncology. and much more. researchers are making unprecedented headway into a wide range of biomedical specialties including immunology. Many researchers are switching to confocal from widefield microscopes or upgrading to a newer confocal technology to get closer to the research answers they’re seeking. Confocal microscopy offers life science researchers a crisp. Choosing the right confocal microscope for your specific research requires careful consideration of the appropriate mix of features related to resolution. sensitivity. Empowered by this technology. clear view into the inner workings of cells. 3 . with novel technology driven by the leading imaging companies. and speed. This greater potential for meaningful discovery goes hand-in-hand with the ability to be published in the most respected peer-reviewed journals. Confocal microscopy has come a very long way since its invention more than a half-century ago. from budgeting to comparing different technologies to asking the right questions of sales representatives. neuroscience. you may want to consider a confocal microscope that will grow with your future needs. and anticipated needs. Evaluate Your Research Needs—Present and Future With such a variety of confocal microscopes on the market.Step 1. customized instrument. Deep tissue Super-resolution Multicolor Live cell Time-lapse Intercellular Intracellular Quantitative imaging 3D imaging You want your investment to last. it's best to evaluate your imaging needs in terms of the “Big Three. high-volume imaging group with very specific imaging needs. your search may lead you to a highly sophisticated. you will likely opt for a product that is very simple to use with a broad range of imaging capabilities. preferences.” 4 . the first step in the buying process is to evaluate your lab’s current areas of research. consider who will be using the imaging system over its life span. Since it is difficult to envision the types of projects you’ll delve into over the next five to 10 years. each providing a unique set of benefits. In addition. But if you’re looking for a confocal microscope to be used by an entire department of researchers with varying levels of expertise. If you are a small. In either situation. or other structures and events on the very small scale. Keeping your primary applications in mind. and how many? How will we split excitation from emission? How many imaging channels will we be working with? Does our research require simultaneous multicolor imaging? The answers to these questions determine the number of Sensitivity laser lines needed for excitation. consider these questions: What are our detection needs? What fluorophores will my lab be using. or focusing on single cells? If you’re seeking to capture interactions between proteins. which beam-splitting device is best. What structures will we be imaging? Will our lab be imaging intracellular or intercellular structures? Will we be mapping tissues and groups of cells. then do we need incubation equipment to keep live cells thriving during an experiment? How much data will our lab be collecting. as with tile scanning experiments. biochemistry. Numerical aperture also comes into play. consider your experimental design. SPEED. and at what rate Speed of acquisition? These questions will help you determine how fast of a scanner you need. 5 Resolution . high-speed imaging is a must and you will want to compare products based on their offerings in this area. What are the dynamics of our experiments? Will our samples be fixed or live? If live. or emerging specialties — different factors will be more important to you than others. the number of detectors needed. optogenetics. super-resolution technology may be required. AND RESOLUTION Depending on your research area — be it neuroscience. including depth and size of the tissues or samples you’ll be imaging. Zoom factor is a meaningful parameter.THE BIG 3: PRIORITIZING SENSITIVITY. To ensure that the confocal hardware can accommodate the resolution your work requires. and whether any specialized detectors are necessary. if you focus on cell biology. but it’s not the only parameter that contributes to resolution. For example. if these features will perform exactly the same whether you add them now or at a later date. However.Step 2. understand that you will be restricted on laser lines and detectors. These advanced models provide greater flexibility for simultaneous multicolor imaging. multicolor images. a modular confocal system is your preferred choice. When comparing models from different companies. A modular confocal also gives you the option of adding advanced techniques such as multiphoton imaging for deep tissue. TIP: If you go with a basic research model. Many people purchase a configurable model with the plan to add upgrades as needed in the future. and it opens the door to more sensitive detectors. TIP: Uncover any limitations of each vendor's configurable platform. 6 . While these basic research models use the same underlying technology as their “big brothers. Here’s a look at the two main classes of products on the market today: Basic Research Model: “A Powerful Start” The lowest cost models fall into this category. you may opt for a system that will move with you as you advance into new research avenues. ask specifically if your desired features will be available in the future.” they have fixed configurations and limited options for upgrades and accessories for live cell imaging. If not. and most importantly. with limited accessories available for live cell imaging. crisp imaging. this model is perfectly acceptable if you are mainly imaging fixed. If you are performing live cell imaging and require advanced spectral capabilities. With these ready-to-use platforms. But some features are not available as upgrades after the time of system purchase. Configurable Model: “Grows With You” The key word here is modularity. A basic confocal will help you easily advance your research from regular fluorescence to clear. Understand the Options There are many confocal microscopes available to match your application requirements and budget needs. even confocal newcomers can quickly produce spectacular 3D. A basic research confocal typically covers routine fluorescence microscopy applications and requires minimal training. sectioned tissues with two or three fluorescent dyes. Multiphoton systems combine advanced infrared lasers and non-descanned detectors for elaborate multicolor multiphoton experiments. many systems offer more than one combination of these features. A favorable option when you’re seeking higher sensitivity than what’s possible with the standard photomultiplier tube. The tunability and flexibility of a spectral detector makes your system ready for new dyes and markers today and in the future. fixed filter. • GaAsP photocathode. A tandem scanner allows you to have two imaging scanners in one system. Captures more light to record the faintest structures from deep tissue sections. but operates with the sample noise characteristics of a standard photomultiplier tube due to traditional dynode amplification. Researchers with high-speed requirements often choose a resonant scanner. DETECTION When evaluating detection systems. Special Accessories for Deep Tissue Imaging When you’re regularly imaging thick specimens such as a living organism or brain tissue. in fact.UPGRADES THAT MATTER Let’s take a look at the most common upgrades to configurable confocal microscopes. • W hite light laser. In general. depending on the samples you’re imaging and the resolution you need. This is the detector choice for multiphoton microscopy. Define your own spectral ranges within the emission spectrum rather than having to use a defined. if you need high resolution. Other common uses for this wavelength include photoactivation or uncaging. and allows for elaborate multicolor. multiphoton experiments. EXCITATION SOURCES • U V laser. SCANNERS • Resonant scanner. these upgrades can be added to your instrument at the time of sale or added later as your research requires or as your budget allows. you can switch between a conventional scanner and a resonant scanner. The resonant scanner can be used to image rapid cellular dynamics. For better signal-to-noise ratio. • Tandem scanner. • Spectral detection. Keep in mind that there are always tradeoffs. and the conventional scanner can be used to capture a large field with image formats up to 8k x 8k. it’s a good idea to weigh the benefits of investing in a mutiphoton confocal instrument. Each confocal brand has a slightly different definition of tandem scanning. plus the additional benefit of pulsed emission. Based on your experimental needs. Especially if deep tissue imaging is central to your research. Allows continuous selection of multiple excitation wavelengths from blue to red through a continuous spectrum. Be aware of this and ask specifically what the tandem scanner does and how it works. That said. The best of both worlds. IR lasers are used for deep tissue imaging since there is less scattering with longer wavelengths. A GaAsP photocathode provides greater sensitivity when imaging dim or weakly stained samples. 7 . the power of today’s confocal technology is quickly making compromise a thing of the past. it is ideal for live cell imaging with rapid experimental dynamics. For example. Need speed? The resonant scanner is an alternative to the conventional scanner that’s usually included in a confocal microscope. • Non-descanned detectors. multiphoton microscopes are the answer. a hybrid detector uses a GaAsP photocathode but offers a lower noise amplification stage. These features are not exclusive of each other. you’ll find that each one offers a variety of special characteristics. you may have to compromise on speed. • Infrared (IR) laser. The 405 nanometer (nm) UV laser is commonly added to image counter stains such as DAPI. Now there’s another option to consider: Resonant scanners. the stage is used to move the sample up or down for focus. In one. On the other hand. and test it out during your product demonstration. and vesicle movement in live cells. superresolution tandem scanner optics resonant scanner 3D imaging infrared laser UV Laser 8 . protein trafficking or interaction. Consider your lab's preferences as you begin the buying process. and the software's features and capabilities are important. but the objective nosepiece moves up and down. a core lab with dozens or more users might desire a static interface that’s extremely easy to use – even for beginners. • A smaller imaging group of confocal experts is more likely to benefit from sophisticated and customized data analysis features. An EM-CCD or sCMOS camera is used for detection. • Upright microscopes allow you to view a specimen from above. be sure to consider what software is available for your application and how easy it is to use. You can image most samples on an inverted microscope that you can on an upright. Resonant Scanning for Fast Live Cell Imaging It used to be assumed that spinning disk technology was the only option for fast live cell imaging. Spinning disk systems have been used historically to image highly dynamic processes such as cell division. the stage is fixed. each with tens of thousands of pinholes. user-friendly workflow. Resonant scanners offer the speed advantage of spinning disk technology with the added benefit of simultaneous multicolor acquisition. • To get an accurate pricing picture. They are most commonly used in applications that involve studying cell cultures in liquid. so be sure that you (and other microscope users) are comfortable with the interface. test a resonant scanner to see whether it will help you accomplish your research goals. Look for an intuitive. So if fast live cell imaging is your priority. or any sample that you do not want to disturb. If you commonly image larger organisms such as a whole mouse. learn what software features are part of the core package versus what’s considered an add-on. with the flat part of a vessel serving as the base. Spinning disk confocal microscopes take a parallel approach to point scanning by using rotating Nipkow disks in the excitation and emission paths. There are two types of upright models. a fixed stage is best. In the other type.OTHER CONSIDERATIONS UPRIGHT OR INVERTED Most confocal microscopes are available in two configurations: upright or inverted. You will be interpreting and managing an immense amount of data. • Most confocal microscopes come with proprietary software. • Inverted microscopes position the imaging objective below the sample. DATA MANAGEMENT — WHAT YOU NEED TO KNOW When evaluating confocal options. lateral resolution isn’t always the most important factor. Here’s a look at the three super-resolution technologies in use today: Localization: A widefield technique that uses thousands of images of stochastically excited fluorophores to generate a super-resolution reconstruction.1 nm Resolution Choosing the best super-resolution system requires an understanding of how each imaging method varies in performance with various sample types. compatible with conventional fluorophores Stimulated Emission Depletion: A point-scanning confocal method that selectively depletes the peripheral region of the diffraction limited scanning spot while leaving a center focal point active to emit fluorescence. Remember. STORM.1 ectr nm on M . Speed and z-resolution are two other key considerations when selecting your super-resolution approach. Benefit: High lateral resolution for fixed samples Structured Illumination: A widefield technique that calculates a super-resolution result from images taken of a sample illuminated with a series of structured masks.1 ic mm ros c op e icr os co pe M icr os co pe s improved the understanding of cellular dynamics at the molecular level. super-resolution microscopy has dramatically H >1 uma mm n E ye C 20 onf 0 n oca m la -1 nd 00 W mm ide fie ld pe r nm -Re . With optical resolution down to 20 nanometers. Known commercially as SIM.2 solu 00 t nm ion M 20 Su El 0. Benefit: Live cell and video rate capabilities (28 frames/second). the answer is yes.IS SUPER-RESOLUTION RIGHT FOR YOU? For many researchers today. Benefit: Simplest transition from traditional light microscopy. no post-processing required 9 . Widefield Microscope Super 1 cm 1 mm 100 µ m 1 µm 100 nm 1 nm 0. Known commercially as STED. and GSDIM. Known commercially as PALM. Budget Your Purchase: A Holistic View When shopping for a confocal microscope. Understand the benefits of modularity. To prioritize your needs. find out what that means. . rank features relative to the experiments you have planned right now. This will allow you to more easily see what you can postpone for a later upgrade versus the features to include at time of sale. if you’re seeking funding approval from within an institution or company. Don’t forget to factor in the service contract. Today’s tougher funding climate means you have to prepare a compelling grant application. and sample preparation costs.. with an eye for systems that can grow with your research by adding new modules catered to your applications. Consider other grant-writing organizations that would support the discoveries you’re pursuing. Seek out the best possible instrument you can afford. can you obtain a special discount? And what specials are offered for core lab managers who own a lot of equipment from the same vendor? TIP: Rank Based on Today’s Needs So you’ve reviewed all of the options and compiled your wish list and. You’ll want to provide the committee with evidence that the instrument will play a central role in solving an important research question. Seek help from the companies who make and sell confocal microscopes. both of which have seen serious budget cuts. you’ll need to show and tell a compelling story about how your new confocal microscope will help your organization accomplish key goals. Likewise. Look at the big picture. Among questions to ask: What is the warranty that comes with the system? How is the service contract structured after the warranty expires? Is there any special pricing offered if you buy multiple years of service contract upfront at the time of sale rather than waiting until the warranty expires? If you stay with a given vendor. 10 Get Creative About Funding Look beyond the National Institutes of Health and the National Science Foundation. To what level is it upgradable? Will it truly grow to meet your needs? Ask to try out an upgraded system so that you can personally attest to the fact it will be suitable for your anticipated research. workflow improvements. Will your new confocal overcome bottlenecks in imaging that currently hinder your lab’s productivity? Are you investing in capabilities you will rarely use? Factor in the productivity improvements. Pay attention to factors such as maintenance. work within your budget and prioritize features based on your most common application needs.Step 3. ask for tips and resources that you can use in your applications. Researchers who must adhere to a limited budget don’t have to sacrifice quality.. as well as the financial impact of being able to obtain images to help achieve your research goals and become published in peer-reviewed journals. Explore the service options and different ways you can buy it. But beware: If a system claims to be modular. you’re over budget. as well as ongoing follow-up to make sure that everything is working as planned. You want to make sure that your vendor support team can easily access your instrument at any time of the day to help resolve urgent problems. Investigate the vendors you’re considering: What is the company's history of innovation? And perhaps most importantly. months. Scientists are always inventing new ways of doing things. weeks. and ask how accessible these experts will be to you if you have a question (and you will likely have lots of questions as you get to know your new system). or even non-urgent ones. and years after the purchase? Here are some questions to ask: What happens after the purchase? You want to do business with a company that will help you get up and running. Learn About Reputation & Support Just as with most significant purchases. and routine maintenance. established community of users. If your lab lacks this internal support. Engage with this community to learn about others’ experiences using the systems you’re considering. not just drop off an instrument at your lab.Step 4. Ask the sales representative whether the company offers extra training or webinars to help keep your knowledge fresh. personalized onboarding process. This is where a company’s strong reputation. Does the company offer field support? Does the company have field representatives who will make a visit to your lab if you need help resolving an equipment problem? What kind of lead time is typical for a visit? Are the company's in-house service experts qualified? Imaging core facilities are often staffed by experts who can help with new user training. 11 . an active community offers the benefit of deep knowledge and innovative ideas. Look for a well defined. Explore the level of scientific expertise the vendor offers in-house. trouble shooting. What are the company's high-tech support capabilities? Remote access is an absolute must today. you’ll want to feel confident in the brand you select. Is there an established community? Look for a group of users who specialize in your key applications. and responsive service support is an advantage. it’s especially important to have a reliable tech support system to call for even simple questions. what level of service will the company offer to its customers in the days. simultaneous laser lines. the extremely narrow reflection band around each selected laser line increases the transmission of emission light. limited by the diffraction of light. a propagating light beam acts as infinite point sources. which can be added to the confocal microscope to provide the speed necessary for real-time imaging of live cells. Unlike fixed dichroic splitters. This fundamental limitation is known as the diffraction limit. this translates into the minimum lateral spacing that can be resolved by an optical system. Dichroic beam splitters. MICROSCOPY TERMS Airy pattern. AOBS is capable of dynamically tuning reflection or suppression of multiple. Point-scanning confocal systems typically use galvanometric mirrors to raster scan the laser across the sample. HyD. which interfere with each other. Airy pattern represents the ideal focused spot of light that a perfect lens with a circular aperture can make. HyDs are capable of photon counting for quantitative imaging applications. This dimensionless number directly relates to the resolving power of a lens. Due to the wave nature of light. Diffraction. Know the Terms: Glossary for Confocal Microscopy As you and your team compare your options. The numerical aperture (NA) of an optical system characterizes the range of angles over which the system can collect or focus light. make sure that everyone is up to speed on the common terminology and how these terms relate to your purchase. A resonant scanner is a special variant of such galvo scanners. Due to this process. 12 . Hybrid Detection (HyD) employs characteristics of both photomultiplier tubes (GaAsP-PMTs) and avalanche photodiodes (APDs) to produce a two-step photodetector capable of vacuum acceleration followed by electron bombardment and avalanche gain. Dichroic splitters used in confocal microscopes can be designed to reflect fixed laser lines (typically one to four lines) and transmit emission spectra to the detectors. Also. Due to the process of diffraction. FEATURES & HARDWARE AOBS. as well as the numerical aperture of the objective used. the resolution of an optical system is limited. The resonant scanner can allow line frequencies up to 24 kHz (as compared to the 3 kHz that non-resonant scanners can achieve). they are used to reflect the excitation light into the sample and transmit emitted fluorescence to the detector. In microscopy. Resonant scanner.Step 5. Lateral resolution. or beam splitters. making it a highly customizable beam splitter. spectrally separate light by transmitting or reflecting light as a function of wavelength. Numerical aperture. It is the product of the sin of the half angle of the cone of light emerging from the objective. In fluorescence microscopy. a beam of light passing through an optical element such as a lens spreads out as it propagates. Diffraction limit. The minimum distance at which two airy patterns of sufficient contrast can be distinguished as distinct objects. Acousto Optical Beam Splitter technology is used in confocal microscopy as a replacement for dichroic beam splitters. The reduced pixel dwell time during resonant scanning reduces photo-stress on fluorochoromes. This is dependent on the wavelength of light used. Dichroic mirrors. and the index of refraction of medium between the lens and sample. METHODS & PLATFORMS CARS. A drawback of widefield microscopy is that fluorescence emitted by the specimen above and below the focal plane interferes with the resolution. this technique enables virtually unlimited resolution. Multiphoton microscopy enables researchers to image deep into thick samples. WLLs emit a continuous spectrum of light between the range of 470 nm to 670 nm. one that excites and the other that de-excites fluorochromes. complements the tunable output of a WLL. Spinning disk systems use many pinholes arranged on rotating disks (or equivalent arrangements). STORM. CARS allows the analysis of samples based on intrinsic vibrational contrast with specificity to molecular bonds within the sample. Coherent Anti-Stokes Raman Scattering (CARS) is a label-free imaging method. Employing two different diffraction patterns. such as an AOBS. GSD works directly on standard fluorophores to achieve super-resolution images with a resolution down to 20 nm. Widefield. are an alternative to conventional lasers used in confocal microscopy. This technique uses laser wavelengths in the infrared to reduce scattering and eliminate out-of-focus fluorophore excitation. A tunable beam splitter. Ground State Depletion (GSD) technology produces 2D and 3D super-resolution images with a precise localization microscopy method. The specimen is generally moved using motorized stages with high precision. Spinning disk. The square pinhole is beneficial because it improves spectral separation by reducing the overlap of colors along a linear detection axis.Square pinhole. labeling steps can be bypassed to image the sample in a minimally invasive manner. which rejects light originating from regions that are out of focus. A super-resolution method that uses combined activator/reporter dye pairs to stochastically achieve super-resolution images with a resolution down to 20 nm. which laser-scans and images the specimen point by point. Since no fluorophores are required. It differs from a confocal. Multiphoton. Supercontinuum lasers. A widefield fluorescence microscope is a conventional type of microscope in which the full field is illuminated and imaged. also know as White Light Lasers (WLLs). compromising the sectioning performance in comparison to true confocal scanning (single point scanning) to increase frame rate. Based on the localization microscopy technique GSDIM (Ground State Depletion microscopy followed by Individual Molecule return). A data set of spatially ordered optical sections that represent a 3D volume. STimulated Emission Depletion Microscopy (STED) is a method that resolves structures below optical resolution and is therefore attributed to super-resolution. Multiple wavelengths can be freely selected to optimize the simultaneous excitation of dye combinations with reduced cross-excitation. An approach for imaging a large specimen by assembling many single images to form one large composite image. Applications range from whole brain imaging to imaging real-time processes within organs. GSD. True confocal laser scanning microscopes focus a single beam on the specimen plane to sequentially point-scan a region of interest with spatial filtration of the emission light through a single pinhole. PALM. Structured Illumination Microscopy (SIM) is a method of super-resolution imaging that uses a modulated illumination pattern and computational restoration to generate a super-resolution image. The geometry of the confocal pinhole determines the diffraction pattern in the intermediate image plane. Tile scanning. Supercontinuum light source (white light laser). SIM. STED. A localization-based super-resolution method that uses photoactivatable fluorophore constructs to stochastically achieve super-resolution images with a resolution down to 20 nm. 13 . Z-stack. Before making a confocal purchase. desired frame rate. desire for 3D images. Contact the manufacturer’s support team to answer any questions that you have. The clearer you can state those goals. Contact the sales representative for the instruments you’re considering. and explain your specific needs. the science is really what you do with the image after it’s been acquired. Don’t forget to try out the software. how many channels. and use your pending purchase as an excuse to make a new connection in your field. become the expert on your new system.Step 6. it’s time to find the perfect match. a demo is to determine whether you can fully • Request a demo. Your next steps: • Reach out to a company salesperson. do some digging to find another researcher who works with similar applications. • Make your decision! After you’ve struck a deal. . Tell the sales rep exactly what you need for the experiment you’d like to perform using the confocal microscope. The goal of questions and learn more. etc. The rep should be able to configure a system and tell you a preliminary price before the demo. Talk to colleagues who use different types of instruments to learn about their experiences: What do they love about their confocal microscope? What do they wish they had considered before their purchase? If you don’t know anyone personally. 14 complete your experiments to your satisfaction. Make a Confident Decision Now that you understand your needs and have evaluated the options. and get active in the community of users. Talk about your sample material. perform working demonstrations with a variety of technologies. how many colors you’ll use. Ask each company to fulfill the same request so that you can compare options with the greatest ease. the better the rep can provide you with the right solution. Formally express your interest in the brand and set an appointment to ask TIP: Be specific or be sorry. Schedule an appointment to ask questions and learn more. workflow improvements. IR lasers). scanners (resonant and tandem). Consider the following: • Sensitivity: What are my detection needs now and in the future? • Speed: What are the dynamics of my experiments? • Resolution: What structures will I be imaging. and detection systems. • Request a demo. this is the right choice. and on what scale? Step 2 Understand the Options • Basic models. • A re the in-house service experts qualified? Seek accessible and knowledgeable application support personnel. and sample prep costs. • Pay attention to maintenance. These models allow greater flexibility for simultaneous multicolor imaging. • Understand the benefits of future upgrades to see how the microscope can grow with your research needs. 15 . as well as those in years to come. • Is field support available? Find out how long it may take to get support when needed. The most common upgrades are excitation sources (UV lasers. (See full glossary in Buyer’s Guide for more. you’ll want to feel confident in the brand you select. but can still produce spectacular images. and non-descanned detection. which offer any combination of options such as spectral. •C  onfigurable models. If you will be performing live cell imaging and require advanced spectral capabilities. Step 1 Evaluate Your Research Needs—Present and Future Evaluate your current research needs. Use this checklist to evaluate your options and guide your purchasing decision. Step 5 Know the Terms Make sure that you are up to speed on common microscopy terms and how they relate to your purchase. • W hat happens after the purchase? You will want help to get up and running. allowing incredible new discoveries. GaAsP photocathode. both virtual and on-site. • Talk to colleagues. • Explore the service options and types of contracts.) Step 6 Make a Confident Decision Do your research and make an informed decision. Step 3 Budget Your Purchase: A Holistic View Prioritize features based on your most common application needs. What do they love about their confocal microscope? What do they wish it could do? • Reach out to a company salesperson. Get your hands on the systems before buying and test your own samples. The lowest cost models have limited upgrade flexibility. Step 4 Learn About Reputation & Support As with any significant purchase.Confocal Microscope Buyer's Guide Checklist: Images captured with today’s confocal microscopes go well beyond what can be seen through a conventional microscope. • Upgrades. com/products/confocal-microscopes/ Copyright © by Leica Microsystems 2014 .www.leica-microsystems.